CN104388301B - Based on unicellular efficient capture device and the system of hydromeehanics - Google Patents

Abstract

The present invention discloses a kind of unicellular efficient capture device based on hydromeehanics and system, wherein, described unicellular efficient capture device comprises: substrate and be arranged on the runner on substrate and arresting structure, wherein, substrate and runner and arresting structure fit together in the way of reversible, wherein, runner and arresting structure comprise multichannel runner, and every road runner comprises cell culture fluid entrance, main flow road, cell capture district and cell culture fluid outlet. The unicellular efficient capture device based on hydromeehanics of the embodiment of the present invention, arrange the cell capture district of continuously series connection on runner, it is to increase space availability ratio, it is to increase unicellular capture rate, and prepare and use cost low, extensibility is strong.

Description

Based on unicellular efficient capture device and the system of hydromeehanics

Technical field

The present invention relates to microfluidic art, particularly relate to a kind of unicellular efficient capture device based on hydromeehanics and system.

Background technology

Research cell when being subject to outside stimulus, how to break up, breed, the characteristic such as die of withering there is the significance in biomedicine. Traditionally, when measuring the characteristic of cell, 1 thousand to 1 hundred ten thousand cell is usually used to count mean value as a colony. But nearest research discovery, the response of cell individual is in fact far from each other with the response of colony, or the mean value saying cell colony masks the real differentiation response of individual cells. Therefore, at biomedical sector, when needing to utilize their response of cell measurement such as aspects such as genetic analysis, medicament research and development, organization formation, cancer mechanism, disease treatments, single cell analysis has had urgent demand.

When doing single cell analysis, sample preparation is an essential step; Not having this step, analysis below cannot carry out. And in sample preparation, the most important catches individual cells one by one the position being namely fixed on and specifying exactly from cell culture fluid, below for convenience of describing, this kind of technology is claimed to be unicellular catching. Because cell must be present in cell culture fluid, therefore the unicellular main stream approach caught is the principle adopting hydromeehanics, and the micro-fluidic chip that design has special construction realizes catching. When realizing unicellular catching, according to whether cell contacts with the bearing surface of surrounding, it is possible to be divided into contact and noncontact formula two kinds. Wherein, noncontact formula method generally adopts stagnant stream or micro-whirlpool to come single particle or cell " suction " center. The mode of stagnant stream that adopts in correlation technique still can not catch general size at 10 ��m of ranks and above cell, adopt micro-vortex manner that the cells quiescent at whirlpool center then can not be made to get off, this can affect the subsides wall performance of cell, is therefore not suitable as the use of subsequent analysis. Therefore, most cells capture device all adopts contact method.

And the contact method in correlation technique is when realizing unicellular catching, there is following shortcoming: the catch position that 1) can not accurately control cell; 2) in the cell culture fluid required for, the sample of cell is enough big, causes the significant wastage of cell sample;3) geometry design of runner is very complicated, adds the preparation requirement to runner and cost; 4) runner design is very long, causes on micro-fluidic chip each effectively to catch unit and is taken up space greatly, causes significant wastage.

Therefore, cell capture device haves much room for improvement.

Summary of the invention

One of technical problem that the present invention is intended to solve in correlation technique at least to a certain extent. For this reason, it is an object of the present invention to propose a kind of unicellular efficient capture device based on hydromeehanics, this capture device arranges the cell capture district of series connection continuously on runner, improve space availability ratio, improve unicellular capture rate, and prepare and use cost low, extensibility is strong.

2nd object of the present invention is to propose a kind of unicellular efficient capture system based on hydromeehanics.

In order to realize above-mentioned purpose, the unicellular efficient capture device based on hydromeehanics of first aspect present invention embodiment, comprise: substrate and be arranged on the runner on described substrate and arresting structure, wherein, described substrate and described runner and arresting structure fit together in the way of reversible, wherein, described runner and arresting structure comprise multichannel runner, and runner described in every road comprises cell culture fluid entrance, main flow road, cell capture district and cell culture fluid outlet.

The unicellular efficient capture device based on hydromeehanics according to embodiments of the present invention, arrange the cell capture district of continuously series connection on runner, it is to increase space availability ratio, it is to increase unicellular capture rate, and prepare and use cost low, extensibility is strong.

In order to realize above-mentioned purpose, the unicellular efficient capture system based on hydromeehanics of second aspect present invention embodiment, comprising: the unicellular efficient capture device based on hydromeehanics of first aspect present invention embodiment; Container, for storing cell culture fluid; And syringe pump, described based on the cell culture fluid entrance in the unicellular efficient capture device of hydromeehanics for described cell cultures liquid pump is entered.

The unicellular efficient capture system based on hydromeehanics according to embodiments of the present invention, arrange the cell capture district of continuously series connection on runner, it is to increase space availability ratio, it is to increase unicellular capture rate, and prepare and use cost low, extensibility is strong.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of unicellular efficient capture device based on hydromeehanics according to an embodiment of the invention;

Fig. 2 A is the schematic diagram that cell flows in runner according to an embodiment of the invention;

Fig. 2 B is the schematic diagram of a road runner and main flow road wherein and trapping region according to an embodiment of the invention;

Fig. 3 be according to an embodiment of the invention in runner corresponding to the volumetric flow rate ratio in each cell capture district during the default value of structure;

Fig. 4 A is that volumetric flow rate ratio is affected schematic diagram (for first cell capture district) by L1, L2, L3 and W1 according to an embodiment of the invention;

Fig. 4 B is that volumetric flow rate ratio is affected schematic diagram (for the 6th cell capture district) by L1, L2, L3 and W1 according to an embodiment of the invention;

Fig. 5 be according to an embodiment of the invention in runner corresponding to the schematic diagram of the volumetric flow rate ratio in each cell capture district during the optimal value of structure;

Fig. 6 is the schematic diagram of the unicellular efficient capture device based on hydromeehanics processed by PDMS soft lithography process;

Fig. 7 A is that cell is schemed by the bright field observed under the microscope after catching according to an embodiment of the invention;

Fig. 7 B is the fluorogram corresponding to bright field figure that cell is observed after catching under the microscope according to an embodiment of the invention;

Fig. 8 is the dynamic schematic diagram of individual cells movement in runner according to an embodiment of the invention;

Fig. 9 is the structural representation of unicellular efficient capture system based on hydromeehanics according to an embodiment of the invention.

Embodiment

Being described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish. It is exemplary below by the embodiment being described with reference to the drawings, it is intended to for explaining the present invention, and limitation of the present invention can not be interpreted as.

Below with reference to the accompanying drawings unicellular efficient capture device based on hydromeehanics and the system of the embodiment of the present invention are described.

Fig. 1 is the schematic diagram of unicellular efficient capture device based on hydromeehanics according to an embodiment of the invention. As shown in Figure 1, the unicellular efficient capture device 10 based on hydromeehanics of the embodiment of the present invention, comprising: substrate 100 and be arranged on the runner on substrate 100 and arresting structure 200.

Wherein, substrate 100 and runner and arresting structure 200 fit together in the way of reversible, and wherein, runner and arresting structure 200 comprise multichannel runner, and every road runner comprises cell culture fluid entrance, main flow road, cell capture district and cell culture fluid outlet.

In one embodiment of the invention, the quantity of the runner that runner and arresting structure 200 comprise can configure according to demand.

In one embodiment of the invention, the cell culture fluid entrance of multichannel runner is separate or be linked to be one piece.

In one embodiment of the invention, the cell culture fluid of multichannel runner exports separate or is linked to be one piece.

Specifically, as shown in Figure 1, the top based on the unicellular efficient capture device 10 of hydromeehanics is divided into runner and arresting structure 200, and bottom is divided into substrate 100. Upper and lower two portions fit together in the way of reversible, can take apart after having caught one batch of cell, and again fitting with new substrate after doing the process such as necessary wash-out, sterilization is used for catching another batch of cell.

Wherein, runner and the arresting structure 200 of upper part comprise multichannel runner, and as shown in Fig. 2 A, Fig. 2 B, each road runner is divided into cell culture fluid entrance, main flow road, cell capture district and cell culture fluid outlet. The quantity of runner can unrestricted choice, and the entrance of these runners can be linked to be one piece or separate existence, and outlet can also be joined together or independent be existed. Original cell suspending liquid imports from entrance, and unnecessary cell culture fluid flows out from outlet.

In one embodiment of the invention, when placement in unicellular efficient capture device 10 horizontal stroke, as shown in Fig. 2 A, Fig. 2 B, the region that every road runner comprises ladder shape or the region that the structure being made up of T font and the font of falling T repeats, wherein, the runner at the upper right corner of each T font or the lower right corner place of the font of falling T narrows from the width, and the region narrowed from the width is cell capture district. In addition, horizontal width runner and vertical wide runner in T font or the font of falling T are called main flow road, allow cell to pass through with cell culture fluid not damaged.

It should be noted that, the unicellular efficient capture device 10 based on hydromeehanics of the embodiment of the present invention, it is possible to have much parallel and that structure is completely the same or different runner. The runner that structure is identical, it is possible to for catching the cell of identical type and size; The runner that structure is different, it is possible to for catching dissimilar and cell that is size. In addition, as shown in Figure 2 A, every road runner all comprises the region of ladder shape, if seen endways for ladder, the left side of the cross bar of ladder or right side are periodically designed with a closing in narrower than runner major part size, the cell that this closes up and near zone is called in the cell culture fluid that cell capture district flows through for catching, other region that channel size is constant is called main flow road, and it allows, and cell culture fluid is accessible to be passed through.

Wherein, the quantity in cell capture district can design as required, and the number of quantity does not affect the unicellular performance caught. In general, 1 square centimeter of yardstick can be arranged up to ten thousand cell capture districts.

In an embodiment of the present invention, each cell capture district has a volumetric flow rate to compare Q₁/Q₂, this value is decided by geometry and the size of runner. For given geometry, taking volumetric flow rate than as objective function, concerning the size of runner, suitable selection should make volumetric flow rate than between 1 and 2. Wherein, the volumetric flow rate in cell capture district compares Q₁/Q₂It is specifically introduced in the embodiment below.

To each road runner, cell is caught by cell capture district one by one in queue mode in cell culture fluid. Each time, in other words, first cell in cell queue is caught by first vacant trapping region, so moves in circles, and all cell capture districts all capture unicellular. On average, unicellular it is about the 1-2 second from entering cell capture district to catching.

Further, the unicellular efficient capture device based on hydromeehanics of the embodiment of the present invention is utilized can to make patterned cellular array. After the cellular array that its making method is to be captured recovers adhesion in substrate 100, two portions up and down of device (i.e. runner and arresting structure 200 and substrate 100) are separated, upper part can reuse and catch cell, and namely leaves patterned cellular array in substrate 100.

In addition, as shown in Figure 1, cell suspending liquid is entered from cell culture fluid inlet pump by syringe pump, after unicellular efficient capture device 10 carries out unicellular catching, unnecessary cell and nutrient solution return to the container that cell suspending liquid is housed from outlet flow, thus save cell suspending liquid.

The unicellular efficient capture device based on hydromeehanics of the embodiment of the present invention, arrange the cell capture district of continuously series connection on runner, it is to increase space availability ratio, it is to increase unicellular capture rate, and prepare and use cost low, extensibility is strong.

As mentioned before, volumetric flow rate compares Q₁/Q₂Value be decided by geometry and the size of runner to be described in detail below.

The present invention utilizes " minimum flow resistance principle " to carry out unicellular catching, according to flow resistance principle, allow cell capture district can catch cell passively, just must the flow resistance in cell capture district be designed lower than the flow resistance in main flow road, and flow resistance height can describe by another convenient parameter, it is exactly volumetric flow rate, it is defined as in the unit time to flow through the volume of the liquid of runner.

Specifically, as shown in Figure 2 A, such as, after fluid enters runner from entrance, there are passage 1 and passage 2 optional time when fluid, if the volumetric flow rate Q of passage 1₁With the volumetric flow rate Q of passage 2₂Ratio Q₁/Q₂Be greater than 1, then fluid will preferentially pass through passage 1, and when so cell flows through with cell culture fluid and closes up 1, cell can be stuck in, by hydrodynamic force, 1 place that closes up, thus realizes catching. Important is, once passage 1 has cell to be stuck in closing in place, this passage can sharply become big because of its flow resistance that gets clogged, substantially exceed the flow resistance in main flow road, therefore cell culture fluid will be selected from main flow road (such as, passage 2 in Fig. 2 A) pass through, thus walk around the closing in that this has captured cell, arrive closing in 2 place of next cell to be captured. The hydrokinetics moved in circles by this kind, after all closing ins place all catch cell, unnecessary cell culture fluid flows out from the exit in main flow road, is collected to use it for anything else. As shown in Figure 2 B, W₂Determine with the principle by coupling cell size of selecting of H numerical value, remaining four variables L₁,L₂,L₃And W₁Then need to select suitable value to make Q₁/Q₂It is greater than 1.Here we assume that cell size to be captured is 15-20 ��m, W₂Less times greater than cell dia to prevent cell culture fluid blocks, one group of value is selected to be: L for=H=25 ��m₁=90 ��m, L₂=6 ��m, L₃=90 ��m, W₁=9 ��m, this group value is claimed to be default value. It is noted that as shown in Fig. 2 A, Fig. 2 B, except closing in place with to mark width be W₁Place, the width of other runner is all W₂, and the height of all runners is all set to H.

The runner of the embodiment of the present invention and the structure of arresting structure 200 have several advantages. The first, space waste is little. Because the runner that we reuse T font and the font of falling T combines, cell capture district can be laid by intensive, highly make use of the change of flow resistance on positive and negative two directions. 2nd, save cell sample. Because cell culture fluid is from ingress to exit, it is all the main flow road of the mode according to parade by mating mutually with cell size; Catching each time, be all that first in queue cell is caught by current cell capture district, in queue, remaining cell all marches to next cell capture district without any damage by main flow road. This kind of deterministic mode of catching determines in theory, has how many cell capture districts, has how many cells just can ensure that all trapping regions complete unicellular catching in cell culture fluid. This is as significant in stem cell and circulation cancer cell to very precious cell sample. 3rd, catch fast, flux height. Because space waste is little, cell culture fluid flows through shorter distance and just reaches trapping region, it means that when catching cell, and speed is faster. In addition, the runner of the present invention and arresting structure 200 (i.e. micro-fluidic chip), it is possible to lay M parallel runner, each runner is laid N number of cell capture district, then flux can reach M and is multiplied by N number of.

Allow the micro-fluidic chip can successful operation, it is necessary to ensure Q₁/Q₂Value be at least greater than 1, this relates to geometrical shape and the size of runner in how this chip of design and optimization. Fig. 2 B marks 6 crucial parameters, wherein the width W in main flow road₂Depend on the cell size to be caught with depth H, can process as a constant in the design, remaining parameter (L₁,L₂,L₃And W₁) then can with Q₁/Q₂For objective function is optimized. Therefore, provide Q below₁/Q₂Expression formula.

According to Darcy-Weisbach equation and Hagen-Poiseuille flow equation solution, the pressure drop of fluid channel can represent and is:

$\mathrm{\Δ}p=\frac{{\mathrm{\ρV}}^2}{2}(f\frac{L}{D}+{\mathrm{\ΣK}}_L),---\left(1\right)$

Wherein, f is darcy friction factor, and L is the length of runner, and �� is fluid density, and V is fluid average flow rate, and D is hydraulic diameter, �� K_LRepresent all because of fluid compression, merging, disperse and the eddy current that causes and the head loss formed.

In an embodiment of the present invention, the maximum reynolds number Re that runner model reaches in an experiment more than 10, can not show that the characteristic of fluid is laminar flow. And for reynolds number Re lower than 10 laminar flow fluid, the pressure drop (first half of formula (1)) caused due to wall friction will much larger than the head loss (latter half of formula (1)) caused due to above-mentioned other reasons, and institute is �� K in (1) with the formula_LIt is negligible. For the runner of rectangular cross section, D can be expressed as 4A/P further, and V can be expressed as Q/A, and wherein A and P is respectively cross-sectional area and the girth of runner, and Q is the volumetric flow rate of runner. Darcy friction factor f is relevant with aspect ratio �� and reynolds number Re=�� VD/ ��, and wherein, �� is hydrodynamic force coefficient of viscosity, and aspect ratio is defined as the ratio of the wide height of flow channel cross-section or width-to-height ratio makes 0�ܦ���1.

For the development laminar flow completely in rectangular cross section runner, it is possible to obtain darcy friction factor f and the relation between aspect ratio �� and reynolds number Re by following formula (2):

C (��)=96 �� (1-1.3553 ��+1.9467 ��²-1.7012��³+0.9564��⁴-0.2537��⁵), (2)

Wherein, C (��) is friction constant, C (��)=(f �� Re)_fd, subscript fd (fullydeveloped) expression develops completely.

And to be formed and to develop laminar flow completely, runner must satisfy condition: L/D > 300. And the runner for the embodiment of the present invention, maximum L/D more than 10, can not develop laminar flow so being not enough to be formed completely. Therefore need, with following formula (3), formula (2) is revised the incomplete development laminar flow conditions being applicable in the present invention, the formula (3) after correction is:

$C=f_{app}\×\mathrm{Re}={\[{\{3.2/{\left(x^{+}\right)}^{0.57})}^2+{(f\×\mathrm{Re})}_{fd}^2\]}^{\frac{1}{2}},---\left(3\right)$

Wherein, x⁺Definition formula is as follows:

x⁺=L/ (D Re)=�� LP²/ (16 �� AQ), formula (2), (3) and (4) are brought in formula (1) and are obtained by (4):

$\mathrm{\Δ}p=\frac{C}{32}\·\frac{{\mathrm{\μlQP}}^2}{A^3},---\left(5\right)$

In one embodiment of the invention, as shown in Figure 2 A, cell culture fluid fluid has (path 1, the first path from one end A in cell capture district to the other end B, by the one of A to B article of straight-line segment) and the 2nd (path 2, path, by the broken line of A-C-D-B), wherein, the pressure drop of the pressure drop of the fluid channel in the first path and the fluid channel in the 2nd path is equal.

In one embodiment of the invention, each cell capture district has respective volumetric flow rate and compares Q₁/Q₂, wherein, Q₁It is the volumetric flow rate in the first path, Q₂It is the volumetric flow rate in the 2nd path, works as Q₁/Q₂When being greater than 1, cell culture fluid fluid preferentially passes through the first path, to catch cell.

Specifically, to two paths 1 from position A to B and path 2, ignoring the trace loss of fluid, formula (5) above can provide the pressure drop on this two paths. The volumetric flow rate in path 1 and 2 as Fig. 2 A indicate. 2nd path (path 2) comprises the first sub-path A-C, the 2nd sub-path C-D and the 3rd sub-path D-B. Pressure drop on path 2 can be described as:

${\mathrm{\Δp}}_2=\frac{{{\mathrm{\μP}}_2}^2}{32{A_2}^3}\·\[2C_{21}{L}_3{Q}_2+C_{22}{L}_1(Q_1+Q_2)\],---\left(6\right)$

Wherein, P₂=2 (W₂+ H), A₂=W₂H, $C_{21}={\[{\{3.2/{\left({x_{21}}^{+}\right)}^{0.57}\}}^2+C{\left({\mathrm{\α}}_2\right)}^2\]}^{\frac{1}{2}},$ $C_{22}={\[{\{3.2/{\left({x_{22}}^{+}\right)}^{0.57}\}}^2+C{\left({\mathrm{\α}}_2\right)}^2\]}^{\frac{1}{2}},$ x₂₁ ⁺=�� L₃P₂ ²/(16��A₂Q₂), x₂₂ ⁺=�� L₁P₂ ²/[16��A₂(Q₁+Q₂)], ��₂=W₂/H��

And for path 1 (the first path), as shown in Figure 2 B, because there are three sections of geometrical shapies in cell capture district, therefore its pressure drop has three parts (i.e. the first sub-path m-n, the 2nd sub-path n-p and the 3rd sub-path p-q) accordingly. For wide and narrow constant part (the first sub-path m-n and the 3rd sub-path p-q), the compute classes of its pressure drop is similar to path 2, and for the arc part (the 2nd sub-path n-p) leniently arriving narrow change, its pressure drop can be expressed as:

${{\mathrm{\Δp}}_1}^{\′}={\∫}_0^{L_T}\frac{C_{12}}{32}\·\frac{{\mathrm{\μQ}}_1{P}^2}{A^3}dL,---\left(7\right)$

Wherein, $P=2(\sqrt{{W_2}^2-4L^2}+H),A=\sqrt{{W_2}^2-4L^2}H,L_T=\sqrt{{W_2}^2-{W_1}^2}/2,$ $C_{12}={\[{\{3.2/{\left({x_{12}}^{+}\right)}^{0.57}\}}^2+C{\left(\mathrm{\α}\right)}^2\]}^{\frac{1}{2}},$ x₁₂ ⁺=�� LP²/(16��AQ₁), $\mathrm{\α}=\sqrt{{W_2}^2-4L^2}/H.$

So, the total pressure drop on path 1 can be expressed as:

${\mathrm{\Δp}}_1=\frac{C_{11}}{32}\frac{\mathrm{\μ}(L_1-L_2-L_T)Q_1{P_2}^2}{{A_2}^3}+\frac{C_{13}}{32}\frac{{\mathrm{\μL}}_2{Q}_1{P_1}^2}{{A_1}^3}+{{\mathrm{\Δp}}_1}^{\′},---\left(8\right)$

Wherein, P₁=2 (W₁+ H), A₁=W₁H, $C_{11}={\[{\{3.2/{\left({x_{11}}^{+}\right)}^{0.57}\}}^2+C{\left({\mathrm{\α}}_2\right)}^2\]}^{\frac{1}{2}},$ $C_{13}={\[{\{3.2/{\left({x_{13}}^{+}\right)}^{0.57}\}}^2+C{\left({\mathrm{\α}}_1\right)}^2\]}^{\frac{1}{2}},$ x₁₁ ⁺=�� (L₁-L₂-L_T)P₂ ²/(16��A₂Q₁), ��₁=W₁/ H, x₁₃ ⁺=�� L₂P₁ ²/(16��A₁Q₁)��

Owing to the pressure drop of fluid channel in the first path is equal with the pressure drop of the fluid channel in the 2nd path, i.e. �� p₁=�� p₂, thus just can obtain Q according to formula (7) and (8)₁/Q₂Solution. Owing to above-mentioned formula is comparatively complicated, it is more difficult to get Q₁/Q₂Analytic solution, it is possible to use software for mathematical computing such as Matlab calculates its numerical solution. In the process calculated, due to Q₁And Q₂Value mutually rely on, therefore by change Q₂(or Q₁) value correspondingly calculate a series of Q₁(or Q₂) value, thus obtain Q₁/Q₂(referring to form 1, form 1 is illustrate at different Q₂Under the Q that obtained by theoretical numerical evaluation₁/Q₂Value). It may be seen that work as Q₂<10^-11m³During/s, no matter Q₂How to change, Q₁/Q₂All substantially, constant, and work as Q₂>10^-11m³During/s, work as Q₂During increase, Q₁/Q₂Can reduce).Work as Q₂<10^-11m³During/s, the maximum difference between C and C (��) is less than 0.6%, therefore has following abbreviation:

C₁₁=C₂₁=C₂₂=C (��₂), C₁₃=C (��₁), C₁₂=C (��).

So, Q is just made₁/Q₂Analytic solution can be obtained as follows:

$\frac{Q_1}{Q_2}=\frac{\frac{C\left({\mathrm{\&alpha;}}_2\right){(W_2+H)}^2(L_1+2L_3)}{{W_2}^3}}{-\frac{C\left({\mathrm{\&alpha;}}_2\right){(W_2+H)}^2(L_2+L_T)}{{W_2}^3}+\frac{C\left({\mathrm{\&alpha;}}_1\right){(W_1+H)}^2{L}_2}{{W_1}^3}+\underset{0}{\overset{L_T}{\&Integral;}}\frac{C\left(\mathrm{\&alpha;}\right){(\sqrt{{W_2}^2-4L^2}+H)}^2}{{\sqrt{{W_2}^2-4L^2}}^3}dL},---\left(9\right)$

Wherein, C (��₁) it is the friction constant in the 3rd sub-path in the first path, C (��₂) it is the friction constant in all sub-paths in the first sub cost sum the 2nd path in the first path, C (��) is the friction constant in the 2nd sub-path in the first path, W₁It is the width in the 3rd sub-path in the first path, W₂Being the width in all sub-paths in the width in the sub-path of first in the first path and the 2nd path, H is the first path and the height in the 2nd path, L₁It is the length in the 2nd sub-path in the 2nd path, L₂It is the length in the 3rd sub-path in the first path, L₃It is first in the 2nd path and the length in the 3rd sub-path, L_TIt is the length in the 2nd sub-path in the first path.

Wherein, it should be noted that the 2nd sub-path (n-p) connecting the first sub-path (m-n) and the 3rd sub-path (p-q) in the first path is made up of the circular arc smoothly transitted.

In formula (9), W₂With H as previously mentioned, numerical value select by coupling cell size principle determine, remaining four variables L₁,L₂,L₃And W₁Then need to select suitable value to make Q₁/Q₂It is greater than 1.

Table 1

These four variables L₁,L₂,L₃And W₁Selection can be determined by the method for Digital Simulation. Simply a bit say, it is possible to use Fluid Mechanics Computation software package, as Fluent calculates the velocity distribution of fluid in runner, and then obtain volumetric flow rate ratio. Here we assume that cell size to be captured is 15-20 ��m, W₂Less times greater than cell dia to prevent cell blocks runner, one group of value is selected to be: L for=H=25 ��m₁=90 ��m, L₂=6 ��m, L₃=90 ��m, W₁=9 ��m. This group value is claimed to be default value. Assuming that each runner has 10 cell capture districts, and not having cell in cell culture fluid, the volumetric flow rate utilizing this group default value to calculate each cell capture district compares as shown in Figure 3. Three representational patterns can be found: the volumetric flow rate of (1) first trapping region is than minimum, and last trapping region is the highest from Fig. 3; (2) the 2nd relative trapping region volumetric flow rates around in trapping regions are than higher; (3) in the middle of, the volumetric flow rate of other trapping region is than relatively stable. Wherein, first and the 3rd meaning bigger. First illustrates that to be paid close attention to first trapping region in the design makes its volumetric flow rate ratio meet the requirements, and the 3rd illustrates that this kind of structure can ad infinitum be expanded and not affect volumetric flow rate ratio or acquisition performance theoretically.

Therefore for determining one group of optimal value, only a certain variable in default value is changed its numerical value below every time, calculating this single variable to the impact of volumetric flow rate ratio, Fig. 4 gives these variablees when changing respectively, corresponding volumetric flow rate than and the variation relation of default value. In fact, the change of any one variable, all trapping regions are all consistent by it by the impact of volumetric flow rate ratio, namely increase simultaneously, or reducing, first trapping region and the 6th trapping region (i.e. representational intermediate capture district) of signal in Fig. 4 A, Fig. 4 B can find out this trend simultaneously. Generally, L₁,L₃And W₁Increase and L₂Reduction can make volumetric flow rate than increase. Finally, through comparing, obtaining one group of optimal value is L₁=120 ��m, L₂=6 ��m, L₃=120 ��m, W₁=10 ��m, the volumetric flow rate of ten corresponding trapping regions is than as shown in Figure 5.

After emulation can verify that current trapping region achieves unicellular catching equally, follow-up first trapping region still can proceed unicellular catching. Being that flow passage structure adopts the aforementioned optimal value obtained shown in form 2, emulation does not have cell capture, first, the 2nd, the 3rd cell capture district be after each captures individual cells respectively, the volumetric flow rate ratio of corresponding all cells trapping region. Visible, after certain trapping region realizes unicellular catching, the volumetric flow rate ratio of this trapping region falls for close to 0 (data referring to having underscore to mark in form 2), losing the suction that fluid produces, thus can not catch multiple cell; Corresponding, the volumetric flow rate of next trapping region declines than also, but still maintains more than 1 (data referring to tilting and add thick mark in form 2). In other words, now next trapping region just becomes first trapping region, and cell capture repeats until all trapping regions all capture unicellular.

Table 2

For the cell of size at 15-20 ��m, one group of design optimization value is L₁=120 ��m, L₂=6 ��m, L₃=120 ��m, W₁=10 ��m, W₂=H=25 ��m is feasible. For the cell that size is different, it is possible to undertaken analyzing by method presented hereinbefore and select.

The preparation of runner and arresting structure 200 can have multiple method. Common comprising uses PDMS soft lithography process, or carries out the micro-processing method such as machining or LIGA with plastics, polymer materials synthetic glass etc., processes runner, closing in, cell culture fluid import and outlet etc. The runner processed and arresting structure 200 tip upside down on substrate 100 carries out plasma body beat oxygen bonding, runner and arresting structure 200 are become the space of permanent closure; Or only carry out vacuum suction after in substrate tipping upside down on, thus form reversible closed flow space. The unicellular efficient capture device 10 this posted keeps flat or makes the higher a little placement in inlet ratio outlet position, is connected with outside fluid path respectively at entrance and exit place. During use, the ratio device that the cell culture fluid being connected with ingress can be placed is high, is added by cell culture fluid fashionable, and by action of gravity, cell culture fluid will flow through runner and trapping region automatically, completes catching of cell.

Fig. 6 show the schematic diagram of runner and the arresting structure 200 processed by PDMS soft lithography process, comprises entrance, runner and outlet. For the object of demonstration, the number of runner only 4 (i.e. M=4) here, in each runner, the number in cell capture district is 100 (i.e. N=100). Drawing attention to, in fact the selection of the number of M and N has very high handiness, it is possible to select as required. Bright field figure and corresponding fluorogram that cell is observed after catching under the microscope it is respectively, it can be seen that the trapping region of 90% has all successfully captured unicellular shown in Fig. 7 A and Fig. 7 B.

Fig. 8 show the dynamic figure that individual cells flows through arrival trapping region, main flow road. Showing the V-bar that individual cells caught is the 1-3 second. And also show cell is observe continuity rule when catching, namely after a cell is caught, caught by next cell capture district to next cell " determinacy ", cell capture district can not be missed, also can not repeat to enter the same trapping region having caught cell. Experimental result also shows, the unicellular power that is captured as is up to nearly 90%.The cell arrangement that these are caught is neat, and its distance can also be changed by the structure of adjusting device. These cells caught can adhere in substrate 100 in original position, adhere to more firm after general 1-2 hour, now runner and arresting structure 200 can be peeled away from substrate 100, just leaving the unicellular array sticking in substrate 100 of patterning, this kind of unicellular array will have purposes very widely.

In order to realize above-described embodiment, the present invention also proposes a kind of unicellular efficient capture system based on hydromeehanics.

Fig. 9 is the structural representation of unicellular efficient capture system based on hydromeehanics according to an embodiment of the invention. As shown in Figure 9, the unicellular efficient capture system based on hydromeehanics of the embodiment of the present invention, comprising: based on the unicellular efficient capture device 10 of hydromeehanics, container 20 and syringe pump 30.

Wherein, container 20 is for storing cell culture fluid (i.e. cell suspending liquid in Fig. 9). Syringe pump 30 is for entering cell cultures liquid pump based on the cell culture fluid entrance in the unicellular efficient capture device 10 of hydromeehanics.

In addition, the outlet based on the unicellular efficient capture device 10 of hydromeehanics is connected with container 20, and the unnecessary cell culture fluid flowed out from the outlet of unicellular efficient capture device 10 flows back to container 20, thus saves cell culture fluid.

The unicellular efficient capture system based on hydromeehanics of the embodiment of the present invention, arrange the cell capture district of continuously series connection on runner, it is to increase space availability ratio, it is to increase unicellular capture rate, and prepare and use cost low, extensibility is strong.

In the description of this specification sheets, at least one embodiment that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to be contained in the present invention in conjunction with concrete feature, structure, material or feature that this embodiment or example describe or example. In this manual, to the schematic representation of above-mentioned term not must for be identical embodiment or example. And, the concrete feature of description, structure, material or feature can combine in one or more embodiment in office or example in an appropriate manner. In addition, when not conflicting, the feature of the different embodiment described in this specification sheets or example and different embodiment or example can be carried out combining and combining by the technician of this area.

In addition, term " first ", " the 2nd " are only for describing object, and can not be interpreted as instruction or hint relative importance or imply the quantity indicating indicated technology feature. Thus, be limited with " first ", the feature of " the 2nd " can express or implicit comprise at least one this feature. In describing the invention, the implication of " multiple " is at least two, such as two, three etc., unless otherwise expressly limited specifically.

Describe and can be understood in schema or in this any process otherwise described or method, represent and comprise one or more for realizing the module of the code of the performed instruction of the step of specific logical function or process, fragment or part, and the scope of the preferred embodiment of the present invention comprises other realization, wherein can not according to order that is shown or that discuss, comprise according to involved function by the mode while of basic or by contrary order, carrying out n-back test, this should be understood by embodiments of the invention person of ordinary skill in the field.

In flow charts represent or in this logic otherwise described and/or step, such as, the sequencing list of the performed instruction for realizing logic function can be considered as, may be embodied in any computer-readable medium, for instruction execution system, device or equipment (as based on system for computer, the system comprising treater or other can from instruction execution system, device or equipment instruction fetch and perform the system of instruction) use, or use in conjunction with these instruction execution systems, device or equipment. With regard to this specification sheets, " computer-readable medium " can be any can comprise, store, communicate, propagate or transmission program for instruction execution system, device or equipment or the device that uses in conjunction with these instruction execution systems, device or equipment. The example more specifically (non-exhaustive list) of computer-readable medium comprises following: the electrical connection section (electronic installation) with one or more wiring, portable computer diskette box (magnetic device), random access memory (RAM), read-only storage (ROM), erasable edit read-only storage (EPROM or dodge speed storer), fiber device, and portable optic disk read-only storage (CDROM). In addition, computer-readable medium is it is even possible that be paper or other the suitable media that can print described program thereon, because can such as by paper or other media be carried out optical scanning, then carry out editing, decipher or carry out process with other suitable methods if desired and electronically obtain described program, then store it in computer memory.

It is to be understood that each several part of the present invention can realize with hardware, software, firmware or their combination. In the above-described embodiment, multiple step or method can realize with the software stored in memory and perform by suitable instruction execution system or firmware. Such as, if realized with hardware, the same with in another enforcement mode, can realize with the arbitrary item in following technology well known in the art or their combination: the discrete logic with the logic gates for data signal being realized logic function, there is the application specific integrated circuit of suitable combinational logic gating circuit, programmable gate array (PGA), field-programmable gate array (FPGA) etc.

Those skilled in the art are appreciated that realizing all or part of step that above-described embodiment method carries is can be completed by the hardware that program carrys out instruction relevant, described program can be stored in a kind of computer-readable recording medium, this program perform time, step comprising embodiment of the method one or a combination set of.

In addition, each functional unit in each embodiment of the present invention can be integrated in a processing module, it is also possible to is that the independent physics of each unit exists, it is also possible to two or more unit are integrated in a module. Above-mentioned integrated module both can adopt the form of hardware to realize, it is also possible to adopts the form of software function module to realize. If described integrated module realize using the form of software function module and as independent production marketing or when using, it is also possible to be stored in a computer read/write memory medium.

The above-mentioned storage media mentioned can be read-only storage, disk or CD etc. Although above it has been shown and described that embodiments of the invention, it is understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, and above-described embodiment can be changed, revises, replace and modification by the those of ordinary skill of this area within the scope of the invention.

Claims (8)

1. the unicellular efficient capture device based on hydromeehanics, it is characterised in that, comprising:

Substrate and be arranged on the runner on described substrate and arresting structure, wherein, described substrate and described runner and arresting structure fit together in the way of reversible, wherein,

Described runner and arresting structure comprise multichannel runner, runner described in every road comprises cell culture fluid entrance, main flow road, cell capture district and cell culture fluid outlet, wherein, the region that described every road runner comprises ladder shape or the region that the structure being made up of T font and the font of falling T repeats, wherein, the runner at the upper right corner of T font described in each or the lower right corner place of the described font of falling T narrows from the width, described in the region that narrows from the width be described cell capture district.

2. as claimed in claim 1 based on the unicellular efficient capture device of hydromeehanics, it is characterised in that, the runner quantity that described runner and arresting structure comprise can configure according to demand.

3. as claimed in claim 1 based on the unicellular efficient capture device of hydromeehanics, it is characterised in that, the cell culture fluid entrance of described multichannel runner is separate or is linked to be one piece.

4. as claimed in claim 1 based on the unicellular efficient capture device of hydromeehanics, it is characterised in that, the cell culture fluid outlet of described multichannel runner is separate or is linked to be one piece.

5. as claimed in claim 1 based on the unicellular efficient capture device of hydromeehanics, it is characterized in that, described cell culture fluid fluid has the first path and the 2nd path from the one end in described cell capture district to the other end, wherein, the fluid-pressure drop in described first path is equal with the fluid-pressure drop in described 2nd path.

6. as claimed in claim 5 based on the unicellular efficient capture device of hydromeehanics, it is characterised in that, cell capture district described in each has respective volumetric flow rate and compares Q₁/Q₂, wherein, Q₁For the volumetric flow rate in described first path, Q₂For the volumetric flow rate in described 2nd path, as described Q₁/Q₂When being greater than 1, described cell culture fluid fluid preferentially passes through described first path, to catch cell.

7. as claimed in claim 6 based on the unicellular efficient capture device of hydromeehanics, it is characterized in that, wherein, described first path comprises the first sub-path, the 2nd sub-path of sub cost sum the 3rd, described 2nd path comprises the first sub-path, the 2nd sub-path of sub cost sum the 3rd, and described volumetric flow rate compares Q₁/Q₂Obtain according to following formula:

$\frac{Q_1}{Q_2}=\frac{\frac{C\left({\mathrm{\&alpha;}}_2\right){(W_2+H)}^2(L_1+2L_3)}{{W_2}^3}}{-\frac{C\left({\mathrm{\&alpha;}}_2\right){(W_2+H)}^2(L_2+L_T)}{{W_2}^3}+\frac{C\left({\mathrm{\&alpha;}}_1\right){(W_1+H)}^2{L}_2}{{W_1}^3}+\underset{0}{\overset{L_T}{\&Integral;}}\frac{C\left(\mathrm{\&alpha;}\right){(\sqrt{{W_2}^2-4L^2}+H)}^2}{{\sqrt{{W_2}^2-4L^2}}^3}dL},$

Wherein, C (��₁) it is the friction constant in the 3rd sub-path in described first path, C (��₂) it is the friction constant in all sub-paths in the 2nd path described in the first sub cost sum in described first path, C (��) is the friction constant in the 2nd sub-path in described first path, W₁For the width in the 3rd sub-path in described first path, W₂For the first sub-path in described first path width and and described 2nd path in the width in all sub-paths, H is described first path and the height in described 2nd path, L₁For the length in the 2nd sub-path in described 2nd path, L₂For the length in the 3rd sub-path in described first path, L₃For the length of first in described 2nd path and the 3rd sub-path, L_TFor the length in the 2nd sub-path in described first path.

8. the unicellular efficient capture system based on hydromeehanics, it is characterised in that, comprising:

The unicellular efficient capture device based on hydromeehanics as according to any one of claim 1-7;

Container, for storing cell culture fluid; And

Syringe pump, described based on the cell culture fluid entrance in the unicellular efficient capture device of hydromeehanics for described cell cultures liquid pump is entered.